Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface.

The fine distribution of the extracellular matrix glycoprotein emilin (previously known as glycoprotein gp115) (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin. 1983. J. Biol. Chem. 258: 13262-13267) has been studied at the ultrastructural level with specific antibodies. In newborn chick aorta the protein was exclusively found within elastic fibers. In both post- and pre-embedding immunolabeling emilin was mainly associated with regions where elastin and microfibrils are in close contact, such as the periphery of the fibers. This localization of emilin in aorta has been confirmed by quantitative evaluation of the distribution of gold particles within elastic fibers. In other tissues, besides being associated with typical elastic fibers, staining for emilin was found in structures lacking amorphous elastin, but where the presence of tropoelastin has been demonstrated by immunoelectron microscopy. This was particularly evident in the oxitalan fibers of the corneal stroma, in the Descemet's membrane, and in the ciliary zonule. Analysis of embryonic aorta revealed the presence of emilin at early stages of elastogenesis, before the appearance of amorphous elastin. Immunofluorescence studies have shown that emilin produced by chick embryo aorta cells in culture is strictly associated with elastin and that the process of elastin deposition is severely altered by the presence of antiemilin antibodies in the culture medium. The name of the protein was derived from its localization at sites where elastin and microfibrils are in proximity (emilin, elastin microfibril interface located protein).

Ultrastructural immuno-localization of tropoelastin in the chick eye.

Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma- of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties.

Hyaluronic acid promotes chick embryo fibroblast and chondroblast expression.

15-day-chick-embryo fibroblasts and chondroblasts were cultured in the presence of high and low molecular weight exogenous hyaluronic acid (HA). Growth range and incorporation of radiolabelled sulphate and proline were determined. HA reduced cell proliferation to about 75% of controls, while incorporation of radiolabelled sulphate and proline was higher in HA-treated cultures of both chondroblasts and fibroblasts. The effect was not due to the polyanionic or polymeric nature of the molecule and appeared to be highly specific for HA.

Changes in the organization of the extracellular matrix in ovarian follicles during the preovulatory phase and atresia. An immunofluorescence study.

The distribution of laminin, type IV collagen and fibronectin was studied by immunofluorescence in rat, pig and cow ovarian follicles. The results obtained in the three species investigated were similar. In all the follicles, laminin and type IV collagen were identically localized in the basal lamina (BL) separating the granulosa and the theca layers. In addition, these two proteins were also distributed in the wall of blood vessels of the thecae and ovarian stroma. The staining showed that the BL of primordial and growing follicles was regular and continuous, but underwent striking modifications during ovulation and atresia. In fact, in preovulatory follicles the BL appeared thinner and discontinuous, whereas it was much thickened and ruptured in atretic follicles. Fibronectin was localized mainly in inner granulosa cells of small and medium-sized growing follicles, and as a broad and irregular layer around the cavity of the degenerated follicles. The results show that each stage of follicular growth and involution is associated with a precise and peculiar pattern of distribution of laminin, type IV collagen and fibronectin. The possibility that these proteins play a role in the local control of ovarian follicular dynamics is advanced.

Fine mapping of tropoelastin-derived components in the aorta of developing chick embryo.

Affinity-purified antitropoelastin antibodies have been used to localize tropoelastin-derived components in aortas from chick embryos of different age by immunoelectron microscopy. Staining in the matrix is first noted at day 3 associated with irregular bundles of filaments resembling microfibrils, in the absence of amorphous elastin deposits. Amorphous material, which rapidly accumulates at later stages, is heavily labelled, while surrounding microfibrils are only poorly labelled. By contrast, a more intense staining of microfibrils persists in regions in which amorphous material is not morphologically evident. These observations indicate that the initial accumulation of elastin requires microfibrils, while the two components are not in close association in the subsequent growth of the amorphous core of the fibre. Intracellular staining is evident in the secretory apparatus of the cell and in peripheral large vesicles. Differentiated cells also show regions of close contact with elastic fibres in which immunological staining for elastin is very close to the cell membrane.

Multiple binding reactivities of an IgG1 mouse monoclonal antibody raised against the extracellular matrix glycoprotein Gp 115.

An IgG1 mouse monoclonal antibody that recognized five different polypeptides from chick and man is described. The antibody was raised against the elastin associated chick glycoprotein gp 115. In addition to the immunizing antigen, antibody 106D6 bound to chick smooth muscle cell myosin and to the alpha, beta and gamma chains of human fibrinogen. Cross reaction was demonstrated by radioimmunobinding, immunofluorescence and immunoblotting experiments. The immunoblotting assays were carried out using identical amounts of the various antigens to exclude the possibility that relevant quantitative differences in their concentration might result in non-specific binding.

Detection of elastin by immunoelectronmicroscopy. A comparison of different procedures.

Elastin components have been identified in chick aorta by different immunoelectronmicroscopic procedures (peroxidase-antiperoxidase, immunoferritin and immunogold) using affinity purified antibodies to chick tropoelastin. The PAP method used in a preembedding procedure stained the outer portion of amorphous elastin and the microfibrils very intensively. The surface of the cells was also slightly stained. On the contrary immunogold labelling on Epon or Lowicryl embedded sections produced a strong decoration only of amorphous elastin, while microfibrils remained almost completely unlabelled. The result is not due to loss of antigenicity of microfibrils during embedding, since similar data were obtained with immunoferritin in a preembedding procedure. Experiments performed under different stringency conditions showed that the products of the peroxidase reaction diffuse and redistribute in the tissue, indicating that the positive staining of microfibrils and cell surface is an artifact. The value of different immunological reagents and procedures in studying the fine mapping of elastin components is discussed.

Relevance of aggregation properties of tropoelastin to the assembly and structure of elastic fibers.

Solutions of tropoelastin incubated under different experimental conditions were examined by electron microscopy after negative staining and after fixation and embedding. Below 37 degrees C only polymorphous structureless elements of variable size could be found. In samples kept for a few minutes at 40 degrees C, flexible, isolated filaments of 5 nm diameter and variable length, together with a few small aggregates of filaments, were seen. No single filaments, but only bundles of filaments were detectable after incubation at 40 degrees C for longer than 5-10 min. Tropoelastin kept at 40 degrees C for longer than 10 hr formed a white precipitate, which, when fixed and embedded as in conventional electron microscopy, consisted of 0.5-2 microns thick, amorphous and branching fibers, identical to those seen in identically processed normal tissues. From these observations a model for the assembly and structure of elastic fibers is proposed.

Monoclonal antibodies against chick gp 115, a matrix glycoprotein with broad distribution.

Hybridoma cell lines were generated producing monoclonal antibodies to chick gp 115, a 115,000-dalton glycoprotein widely distributed in the connective tissue. The specificity of the antibodies was determined by indirect radioimmunobinding: the extent of binding was a function of i) antigen and ii) antibody concentration; iii) inhibition of binding of radiolabelled antibody by unlabelled antibody and iv) among many known extracellular collagenous or noncollagenous glycoproteins tested only gp 115 gave a strong positive binding reaction. The antibodies were used for indirect immunofluorescence and a strong staining reaction was detected in all blood vessels, around smooth muscle cells in several organs, and in the connective matrix of other tissues such as the liver, and the lung. Based on the competition of binding of [125I]-labeled purified antibody by unlabeled antibodies, two separate epitopes were identified on gp 115. Further analysis of the localization of the epitope was obtained by CNBr cleavage and partial digestion of gp 115 with Staphylococcus aureus V8 protease and alpha-chymotrypsin digestion. Following CNBr cleavage a major fragment of Mr = 35,000 was recognized by 4 monoclonal antibodies, and fragments of comparable Mr were detected following V8 protease and alpha-chymotrypsin digestion.

Glycoprotein 115, a glycoprotein isolated from chick blood vessels, is widely distributed in connective tissue.

An extracellular glycoprotein (gp 115) with an apparent Mr = 115,000 isolated from chick aortas (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin, 1983, J. Biol. Chem., 258:13262-13267), was used to immunize mice. The antisera were shown to specifically recognize gp 115 by numerous criteria: a major band around Mr = 115,000 plus minor bands of lower Mr were visible by immunoblotting on aorta extracts, and a similar pattern was observed with a monoclonal antibody; no cross-reactivity was detected by radioimmunobinding with other extracellular proteins, namely, fibronectin, laminin, and collagen types I, III, IV, V, and VI. Antigen distribution on frozen tissue sections from newborn chicks was investigated by using affinity-purified antibody. Strong immunoreactivity was always found in blood vessels. In the digestive tract, the fluorescent staining was localized both at the level of muscular layers and in the stromal matrix of the villi. Within skeletal muscle and myocardium, staining was associated with large connective tissue bundles and the matrix around each muscle fiber. Intense fluorescence was observed in the kidney, in smooth muscle cells rich areas of parabronchi, and within the portal space and along liver sinusoids. The antigen was not detected at the epidermal-dermal junction; immunoreactivity in the dermis was present as a diffuse fibrillar pattern. That the antigen detected by immunofluorescence in the various organs was indeed gp 115 was demonstrated by immunoblotting analysis: as in aorta extracts, a major band around Mr = 115,000 was detected in several tissues. Antibody-reacting material was also incorporated into the extracellular matrix produced by embryo smooth muscle cells grown in vitro and was organized as a meshwork of fine fibrils.

Elastin fiber-associated glycosaminoglycans in beta-aminopropionitrile-induced lathyrism.

Ruthenium red and toluidine blue O precipitates were described associated with lathyritic elastic fibers in aortas of chickens treated with beta-aminopropionitrile fumarate (I. Pasquali-Ronchetti, C. Fornieri, I. Castellani, G. M. Bressan, and D. Volpin (1981). Alterations of the connective tissue components induced by beta-aminopropionitrile. Exp. Mol. Pathol. 35, 42-56). In this report evidence is given that these precipitates reveal the presence of proteoglycans, as they are completely removed by 5 M guanidine-HCl incubation and by specific enzymatic digestions. In particular, proteoglycans associated with the poorly cross-linked lathyritic elastin can be removed by testicular hyaluronidase, chondroitinase ABC, heparitinase, and nitrous acid treatments, whereas they are rather resistant to streptococcal hyaluronidase and chondroitinase AC. On the contrary, proteoglycans of the matrix or associated with collagen fibers are particularly sensitive to these latter enzymatic treatments. The conclusion is reached that glycosaminoglycans associated with beta-aminopropionitrile-induced lathyritic elastin (i) are different from those of the matrix or associated with collagen, and (ii) include mainly dermatan and heparan sulfates.

Isolation and characterization of a 115,000-dalton matrix-associated glycoprotein from chick aorta.

Chick aortas were extracted sequentially with phosphate-buffered saline, 6 M guanidine HCl, and 6 M guanidine HCl containing dithioerythritol. The proteins present in the guanidine HCl + dithioerythritol extract were separated by DEAE-cellulose chromatography, and the fractions recovered were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five major glycoprotein components with apparent Mr = 205,000, 195,000, 150,000, 135,000, and 115,000 (gp 115) were identified. gp 115 was further studied since it was the only noncollagenous protein based on amino acid analysis. The protein was purified to homogeneity by preparative electrophoresis. Its amino acid composition was characterized by a high content of glutamic acid and arginine and a relatively high content of leucine, glycine, and alanine. The concentration of gp 115 in the guanidine HCl + dithioerythritol extract was about 15-fold that in the guanidine and saline extracts. Overall, about 80% of the protein was solubilized with guanidine HCl + dithioerythritol, suggesting that most of it formed large aggregates stabilized by disulfide bonds in vivo. Immunofluorescence studies with specific antibodies showed that gp 115 formed an extracellular fibrillar network in the aorta wall. One-dimensional finger printing with Staphylococcus aureus V8 protease and immunological studies indicated that the protein was unrelated to fibronectin and laminin. The data led us to conclude that gp 115 is a novel extracellular component of chick aorta.

Banded fibers in tropoelastin coacervates at physiological temperatures.

Tropoelastin was purified from aortas of chicks grown on a beta-aminopropionitrile-containing diet. The preparation could be considered pure following the criteria of amino acid composition and gel electrophoresis. When aqueous solutions of tropoelastin (5 mg/ml) were warmed to 40 degrees C (physiological temperature for chicken) for 10 min, and observed by negative-staining electron microscopy, it revealed the presence of two kinds of ordered structures. One consisted of densely packed parallel filaments with a center-to-center distance of about 5 nm, and the other of banded fibers, 100-150 nm in diameter, with a cross periodicity of about 55 nm. In some areas the fibers appeared to be formed by lateral aggregation of 1.5-2-nm-thick microfilaments. The fibers were similar to those previously obtained with the synthetic polypentapeptide of elastin (Val-Pro-Gly-Val-Gly)n and degradation products of elastin at temperatures much higher than the physiological one. The results indicate that the property of tropoelastin to form ordered structures is intrinsic to some of the polypeptide sequences of the molecule and that hydrophobic forces are involved in the formation of the aggregates.

Chemical characterization of the hen eggshell matrix: isolation of an alkali-resistant peptide.

The eggshell matrix was obtained from hen eggshells using EDTA solutions. The water-soluble organic material was separated on a DEAE-cellulose column equilibrated with 8 M urea using Tris-hydrochloric acid buffer as eluent with a linear gradient of sodium chloride. Five main fractions were obtained which differ in amino acid composition and sugar contents. As is shown from the uronic acid content, the first two fractions eluted from the column are glycoproteins, while the other three contain proteins and glycosaminoglycans From the alkaline hydrolysate of the eggshell matrix, a peptide was isolated which is composed of aspartic acid, threonine, serine, glutamic acid, proline, glycine and alanine in a molar ratio of 2:1:3:7:1:3:1 with a minimum molecular weight of 2158 daltons. The calcium ion binding to this peptide was studied, at different pH values, with both free and blocked carboxyl groups, using murexide as an indicator of free Ca2+. The importance of this acidic peptide in the calcification process of the eggshell matrix is discussed.

Elastin and collagen in the normal and obstructed urinary bladder.

Histological and chemical studies were carried out on the elastin and collagen content of the normal and the obstructed urinary bladder wall. The observations were made on 3 different age groups: (1) 3-month-old children with vesical outlet obstruction, (2) children aged between 4 and 8 years with partial vesical obstruction, and (3) adults with a chronically obstructed bladder. An increase in elastic tissue was shown only in the newborn and in the adults. The collagen did not vary in any of the cases studied. The elastin showed an increase in polar amino acids in the pathological organs compared with the normal. A similar increase in elastin was observed in normal individuals as a result of the ageing process.

Pseudoxanthoma elasticum. Biochemical and ultrastructural studies.

Ultrastructural and biochemical studies of the dermis from patients with pseudoxanthoma elasticum are described. Elastin fibers showed mineral deposits that were associated with an elastin material exhibiting altered affinity for the electron microscopical stains and altered ultrastructural organization. In the most affected patient the elastin was shown to be increased and to have an abnormal amino acid composition. In some patients the collagen fibers were laterally fused. The ground substance was increased as evidenced by electron microscopy and confirmed by a slight increase in uronic acids, hexosamines, and neutral sugars.

Some observations on the calcium ion binding to the eggshell matrix.

The calcified matrix of the hen eggshell has been demineralized with the EDTA. Aliquots of this material are soluble in water and have been characterized by column chromatography and by chemical analyses. Of particular interest is the high hexosamine and uronic acid content, which confirms the protein-polysaccharide nature of this water-soluble material. The calcium ion binding to the eggshell matrix has been studied by the equilibrium dialysis technique at different pH values, with both free and blocked carboxylic groups. The material with the free carboxylic side chain groups binds more calcium ions with increasing pH value. When the carboxylic groups have been previously blocked with a water-soluble carbodiimide, the calcium ion binding rapidly decreases. The residual capacity to bind calcium ions in the material with the carboxylic functions modified is probably due to the sulfate ions. In agreement with previous observations on other calcified substrates, the calcium ion binding seems to depend on the presence of ionized carboxylic functions of the matrix.

Ultrastructural and biochemical studies on a case of elastosis perforans serpiginosa.

Ultrastructural and biochemical studies on the collagen and elastin fibres of the dermis from a patient with Elastosis perforans serpiginosa are described. Qualitative and quantitative alterations on collagen and elastin are shown, which give some evidence for considering the disease as a connective tissue defect.

Age-related changes in the reducible cross-links of human dermis collagen.

Samples of normal human dermis of different ages are reduced with tritiated sodium borohydride and changes of major reducible cross-links are compared as a function of chronological age. While lysinorleucine practically remains constant, reduced desmosine changes slightly, hydroxylysinoroleucine and dihydroxylysinonorleucine display a marked decrease with age. An unknown compound is shown to increase with aging. The data suggest a correlation between the change of aldimine cross-links and the structural and/or biochemical changes occurring with increase in age.